PROJECT SUMMARY/ABSTRACT Many drugs pass through preclinical and early clinical studies before safety concerns are realized, putting patients at risk and creating a bottleneck in the drug development process. The long-term objective of our research is to improve human risk assessment in drug safety testing. Genetic susceptibility is an important feature of adverse drug reactions not currently represented in preclinical toxicology models. Therefore, we hypothesize that controlled incorporation of genetic diversity in preclinical safety studies would improve prediction and understanding of adverse drug reactions in humans. Furthermore, the identification of specific genes and pathways contributing to toxicity susceptibility would allow us to better understand the relationship between preclinical toxicology findings and patient response. We have previously demonstrated the utility of the Collaborative Cross (CC) mouse population to model toxicity responses that require genetic susceptibility factors. Currently, the CC approach requires large in vivo studies that are time consuming, expensive, and limited in scope. We are developing a novel in vitro CC platform containing primary cells isolated from CC lines and cultured on multi-well plates to allow for multiple concentrations, treatment regimens, and endpoints to be assayed across replicate wells in a single experiment. Our platform will enable the rapid and cost-effective identification of gene-by-treatment interactions associated with adverse drug response at all stages of drug development. We are beginning platform development with cultured CC hepatocytes. This will support an initial focus on drug- induced liver injury (DILI), which is one of the main adverse responses leading to the termination of clinical drug development programs and withdrawal of approved drugs from the market, and an area in which we have well- established expertise. The platform will include cryopreserved hepatocytes isolated from CC lines and cultured in 3D spheroids which will increase the physiological relevance of the in vitro model while decreasing the number of cells (and animals) needed overall. We will evaluate the utility of the in vitro CC platform to screen new drug candidates for DILI liability and aid in the improved estimation of maximum safe starting dose for first-in-human clinical trials (Aim 1); provide new understanding of the mechanisms of DILI and inform precision medicine risk mitigation strategies to improve patient safety and reduce the cost of drug development (Aim 2); and validate causal associations and inform species differences in genetic factors contributing to drug response (Aim 3). Collectively, our proposed research will improve preclinical drug safety screening and support the identification of genetic risk factors, mechanisms, and interspecies differences contributing to drug toxicity in humans. Together, these insights will further reduce the potential for patient harm and the cost of drug development.